1. Field of the Invention
This invention relates to yeast protein secretion technology. More specifically, it relates to recombinant Yarrowia lipolytica cloning vehicles comprising heterologous DNA coding for expression and secretion of mammalian protein (e.g., prochymosin) and other polypeptides; and to expression vectors comprising a Y. lipolytica gene promoter (e.g. XPR2 or LEU2), alkaline protease singal (or pre) sequence, pro region, and XPR2 terminator region, and variants or functional equivalents thereof arising from degeneracy of the genetic code or use of other Y. lipolytica gene component. Additionally, it relates to yeast transformants carrying said expression and secretion vectors, their use to produce heterologous proteins in their native, functional state; and methods for accomplishing the above.
2. Description of the Prior Art
The economic attractiveness of a steady and sufficient supply of a variety of proteins or polypeptides valuable to an industry (e.g., prorennin, bovine growth hormone) or for medicinal purposes (e.g., urogastrone, tissue plasminogen activator, human anaphylatoxin C5a) and particularly of a source which affords high quality product in an easily recoverable, functional form has led many investigators to apply recombinant DNA technology to microorganisms as "factories" for production of heterologous proteins.
Extensive research is focussed on protein secretion as a potential solution to difficulties encountered in recovering exogenous or heterologous (foreign) protein in a biologically active form from intracellular accumulations in recombinant host cells, especially from Escherichia coli. In E. coli, the heterologous protein is often produced within the cell in the form of refractile inclusion bodies. Said protein is generally of low water solubility and has little or no biological activity. Extraction of said protein from the refractile inclusion bodies generally involves harsh chemical treatment which may be costly and can result in little or no recovery of the protein in the desired, native, biologically active form. Further, the possibility of contamination of said protein with undesirable substances produced by E. coli is aggravated by the need to disrupt the cells in order to release the refractile bodies. Other organisms besides E. coli also produce heterologous protein in insoluble intracellular form. For instance, British Patent No. 2,091,271, published July 28, 1982, discloses genetic modification of S. cerevisiae via recombinant DNA technology to express calf rennin, or chymosin, the terms are used interchangeably herein. In view of these difficulties secretion of said protein from the host organism has been turned to in an attempt to produce the protein in a native, active configuration.
Whether a particular protein, including heterologous protein, or polypeptide is secreted by a given organism appears to be dependent upon the protein. In most eucaryotic cells, some of the protein synthesis apparatus is associated with the endoplasmic reticulum membrane and the sequence of amino acids (called the "signal sequence") near the amino-terminus of the nascent polypeptide chain serves to direct the protein to cross the membrane. The signal sequence is subsequently cleaved proteolytically during the secretion process affording active, mature protein. Several attempts have been made to develop processes for secreting heterologous proteins using signal sequences in microorganisms, including Bacillus subtilis, Saccharomyces cerevisiae and in mammalian cells in culture. However, said organisms have not proven to be ideal.
Inherent properties of B. subtilis, e.g., secretes many proteins including numerous proteases which tend to degrade the secreted heterologous protein; instability of transformed strains resulting from the loss of heterologous DNA, have hindered its development.
Mammalian cells have been successfully genetically engineered to express and secrete heterologous proteins, but these systems are technologically demanding and expensive to operate and remain impractical for commercial production of most proteins as products.
While protein secretion studies have been more successful with S. cerevisiae than with B. subtilis, even S. cerevisiae appears to have some inherent limitations as a protein secretion system. European Patent Application No. 0123544, published Oct. 31, 1984, describes isolation of the S. cerevisiae alpha-factor genes, and use of the promoter and/or signal peptide portions thereof in combination with DNA coding for proteins heterologous to yeast in a plasmid for transformation of yeast cells capable of producing discrete, mature protein upon cell culture. EP Application No. 0088632, published Sept. 14, 1983, describes a process for expressing and secreting heterologous protein in S. cerevisiae. However, the size of the proteins which S. cerevisiae will efficiently secrete with these and other secretion systems appears to be limited to about 20,000 daltons. Overcoming this general inefficiency of S. cerevisiae as a secretion organism has required multiple mutational alterations as described by Smith et al., Science 229; 1219-1224 (1985). One exception to this trend is the observation that Aspergillus enzymes larger than 20,000 apparently can be secreted by S. cerevisiae, but these enzymes are highly glycosylated by S. cerevisiae and this may influence the efficiency of secretion.
Particular interest resides in Yarrowia lipolytica, an industrially important species of yeast used to produce citric acid and single cell protein. It can also be used to produce erythritol, mannitol and isopropylmalic acid. In contrast to S. cerevisiae, Y. lipolytica is of special interest and value because of its ability to efficiently secrete high molecular weight proteins (alkaline protease, acid protease and RNAse) into its growth medium thus permitting potential recovery of heterologous proteins in the native state without the need of disrupting the producing cells. Additionally, Y. lipolytica secretes very few proteins in quantity thus offering potential for production of a desired heterologous protein in the growth medium as the predominant protein species and simplifying recovery of said heterologous protein product.
Y. lipolytica produces high levels of extracellular protease. This is the predominant protein secreted by Y. lipolytica. The particular protease (alkaline, acid or neutral) depends upon the strain of Y. lipolytica used (Ogrydziak et al., J. Gen. Microbiol. (1982) 128, 1225-1234). A partial sequence analysis of the N-terminal amino acid sequence of alkaline extracellular protease is reported by Ogrydziak et al., (loc. cit.).
Copending application Serial No. 634,505, filed July 25, 1984, describes methods for transforming Y. lipolytica and for cloning Y. lipolytica genes by complementation of mutations. It discloses the cloning of the XPR2 gene, which codes for a secreted alkaline protease, by complementation of an xpr2 mutation of Y. lipolytica. The methodology includes transforming a host strain of Y. lipolytica with a BglII partial digest of a Y. lipolytica gene library in the vector pLD40 described in EP application No. 0138508, published Apr. 24, 1985, the counterpart of the above-identified U.S. application.